The ability to see and track single molecules inside living cells would be invaluable for reconstructing the sequential location-specific interactions of proteins characteristic of signal transduction pathways, as many proteins involved in signal transduction shuttle between spatially distinct regions of the cell such as the nucleus and the plasma membrane. It is possible to track briefly single molecules inside cells using chemical fluorophores, specialized microscope setups and extremely intense illumination. However, bleaching of the chemical fluorophore sets an upper limit of several seconds for time of observation. As a result, the technical difficulty of this powerful technique is such that it is limited to specialized biophysics laboratories. We hypothesize that quantum dots could serve as the key enabling technology to allow cell biologists and cancer researchers to apply single molecule methods to the study of signal transduction. Quantum dots are intensely fluorescent semiconducting nanocrystals. They are similar in size to GFP molecules (2-10 nm). Single quantum dots are visible by either digital detector or by eye through a standard research grade microscope and do not photobleach. As quantum dot technology is extremely new, their use as probes for tracking single molecules in living cells has not yet been explored. The specific aims of this proposal are: 1) To devise techniques for the creation and validation of single molecule protein/quantum dot conjugates that can be introduced into cells; and 2) to conduct a series of feasibility studies of quantum dot conjugates as single molecule probes inside living cells. We will test both direct techniques in which conjugates are directly linked with quantum dots and indirect techniques in which the quantum dots are conjugated to fab fragments of monoclonal antibodies directed against peptides or epitope tags on the proteins of interest. The usefulness of these conjugates will be tested first as probes that can be added to the cell surface (e.g. the endosomal marker transferrin), and then in pilot studies of cytoplasmic proteins that are important members of signal transduction pathways important to cancer development (e.g. grb2 and p53). If these studies show promise, we will initiate more extensive studies since understanding of signal transduction pathways can translate directly into pharmacological interventions aimed at treating human cancer. [unreadable] [unreadable]